Mycoplasma agalactiae Induces Cytopathic Effects in Infected Cells Cultured In Vitro

Abstract

Mycoplasma agalactiae is the etiological agent of the contagious agalactia syndrome in sheep and goats and causes significant economic losses worldwide. Yet the mechanism of pathogenesis is largely unknown. Even whole-genome sequence analysis of its pathogenic type strain did not lead to any conclusions regarding its virulence or pathogenicity factors. Although inflammation and tissue destruction at the local site of M. agalactiae infection are largely considered as effects of the host immune response, the direct effect of the agent on host cells is not completely understood. The aim of this study was to investigate the effect of M. agalactiae infection on the quality and viability of host cells in vitro. Changes in cell morphology including cell elongation, cytoplasm shrinkage and membrane blebbing were observed in infected HeLa cells. Chromatin condensation and increased caspase-3 cleavage in infected HeLa cells 48 h after infection suggests an apoptosis-like phenomenon in M. agalactiae-infected cells. In compliance with these results, decreased viability and cell lysis of M. agalactiae-infected HeLa cells was also observed. Measurement of the amount of LDH released after M. agalactiae infection revealed a time- and dose-dependent increase in HeLa cell lysis. A significant decrease in LDH released after gentamicin treatment of infected cells confirmed the major role of cytadherent M. agalactiae in inducing host cell lysis. This is the first study illustrating M. agalactiae's induction of cytopathic effects in infected HeLa cells. Further detailed investigation of infected host tissue for apoptotic markers might demonstrate the association between M. agalactiae-induced host cell lysis and the tissue destruction observed during M. agalactiae natural infection.

Fig 1. Percentage Viabilty of M. agalactiae-infected HeLa cells.

Viable HeLa cells were measured using Trypan blue staining at 24, 48 and 72 h p.i. Mean values ± SD from three independent experiments performed in duplicates are depicted.

Fig 2. Phase contrast microscopic images showing the morphological changes in M. agalactiae-infected cells.

Images were taken at 24, 48 and 72 h p.i. at 10x magnification. Pointed arrows show the profound cell elongation and membrane blebbing in infected HeLa cells.

Fig 3. DNA fluorescence staining showing chromatin condensation in M. agalactiae-infected HeLa cells 48 h p.i.

Cells were permeabilized and stained with DAPI, and images were taken using a fluorescence microscope. As a positive control, HeLa cells were treated with 1 μM staurosporine for 3 h and stained with DAPI. Bars, 10 μm.

Fig 4. Caspase-3 cleavage in M. agalactiae-infected HeLa cells.

The graph represents the average relative density of cleaved caspase-3 from the immunoblots of M. agalactiae-infected (+ MA) and uninfected (- MA) HeLa cells at 24 and 48 h. As a positive control, HeLa cells were treated with 1 μM staurosporine (Sp) for 3 h. Experiments were performed at least three times, and average relative density values ± standard deviation are represented here. ‘*’ indicates p < 0.05.

Fig 5. Percentage of cell lysis in M. agalactiae-infected HeLa cells.

Both time- and dose- dependent increase (p<0.05) was observed at 36 and 48 h p.i. with all tested MOIs compared to uninfected cells in absence of gentamicin. A significant decrease in cell lysis was observed in gentamicin-treated cells (+G) compared to gentamicin-untreated cells (-G) at 48 h p.i. (‘*’ indicates p<0.05).